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BIOACTIVITY AND CHEMICAL INVESTIGATIONS OF PERESKIA BLEO AND PERESKIA GRANDIFOLIA

SIM KAE SHIN

THESIS SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

FACULTY OF SCIENCE UNIVERSITY OF MALAYA

KUALA LUMPUR

2010

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UNIVERSITI MALAYA

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ii BIOACTIVITY AND CHEMICAL INVESTIGATIONS OF

PERESKIA BLEO AND PERESKIA GRANDIFOLIA

ABSTRACT

Ethnopharmacological data has been one of the common useful criteria in drug discovery. Pereskia bleo and Pereskia grandifolia (Cactaceae), commonly known as

‘Jarum Tujuh Bilah’ in Malay and ‘Cak Sing Cam’ in Chinese have long been used as natural remedies in Malaysia. The experimental approach in the present study was based on bioassay-guided fractionation. The crude methanol and fractionated extracts of both Pereskia spp. were initially investigated for their biological activities such as antioxidant, antimicrobial and cytotoxic effect against five human cancer cell lines, namely nasopharyngeal epidermoid carcinoma cell line (KB), cervical carcinoma cell line (CasKi), colon carcinoma cell line (HCT 116), hormone-dependent breast carcinoma cell line (MCF7), lung carcinoma cell line (A549) and the non-cancer human fibroblast cell line (MRC-5) using in vitro cytotoxicity assay, in order to identify the bioactive extracts of both Pereskia spp.

The hexane and ethyl acetate extracts of both Pereskia spp. generally showed stronger antioxidant activities than the other extracts. Ethyl acetate extracts of both Pereskia spp. also showed some mild antimicrobial activities against the tested bacteria.

In the cytotoxicity assay, both Pereskia spp. exerted no damage to MRC-5 normal cells.

The ethyl acetate extracts of both Pereskia spp. in general gave higher inhibition and stimulation values against various cancerous cell lines compared to other extracts. The cell deaths of the selected cancer cells elicited by the cytotoxic active extracts of both Pereskia spp. were found to be apoptotic in nature based on a clear indication of DNA fragmentation, which is a hallmark of apoptosis. In addition, the LUX RT-qPCR [real-

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iii time reverse transcriptase–polymerase chain reaction (RT-qPCR) using LUX (Light Upon eXtension) primers] analysis showed that apoptosis elicited by the cytotoxic extracts on selected cancer cells was mediated by p53, caspase-3 and c-myc activation in different expression levels.

Methyl palmitate, methyl linoleate, methyl α-linolenate and phytol were identified from the hexane extract of Pereskia bleo by GCMS analysis whilst methyl palmitate, methyl linoleate, methyl α-linolenate and methyl stearate were identified from the hexane extract of Pereskia grandifolia. From the results of the biological screenings, it is observed that the ethyl acetate extracts generally have stronger biological activities than other extracts. Further chemical investigations were thus directed to the ethyl acetate extracts of both Pereskia spp.

2,4-Di-tert-butylphenol (1), α-tocopherol (2), phytol (3), ß-sitosterol (4), dihydroactinidiolide (5) and a mixture of sterols containing campesterol (6), stigmasterol (7) and β-sitosterol (4) were isolated from Pereskia bleo whilst 2,4-di-tert- butylphenol (1), α-tocopherol (2), ß-sitosterol (4) and mixture containing methyl palmitate (9), methyl oleate (10), methyl stearate (11) and 2,4-di-tert-butylphenol (1) were isolated from Pereskia grandifolia. It is interesting to note that 2,4-ditert- butylphenol (1), α-tocopherol (2) and ß-sitosterol (4) were isolated from the ethyl acetate extracts of both Pereskia spp. The cytotoxic activities of the isolated constituents were evaluated against the above human cell lines and further studies on their mode of action suggested that these activities are connected with induction of apoptosis.

In addition, the toxicity of both Pereskia spp. was evaluated in vivo and were considered safe in acute oral toxicities in experimental mice. The screening of the

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iv locally grown Pereskia bleo and Pereskia grandifolia indicated the presence of alkaloids, but the concentration was very low.

The findings of Pereskia bleo and Pereskia grandifolia in the present study provided scientific validation on the use of the leaves of both Pereskia spp. for the treatment of cancer. Further studies on the mutagenic and toxicity effect over a longer period of time involving detection of effects on vital organ functions should be carried out to ensure that the plants are safe for human consumption.

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v KAJIAN BIOAKTIVITI DAN KIMIA

PERESKIA BLEO DAN PERESKIA GRANDIFOLIA

ABSTRAK

Data etnofarmakologi merupakan salah satu kriteria umum yang berguna dalam penemuan sesuatu ubat tertentu. Pereskia bleo dan Pereskia grandifolia (Cactaceae) yang biasa dikenali sebagai ‘Jarum Tujuh Bilah’ di kalangan orang Melayu dan ‘Cak Sing Cam’ di kalangan orang Cina telah lama digunakan sebagai jamu semulajadi di Malaysia. Eksperimen dalam penyelidikan ini dijalankan berdasarkan pemfraksian berpanduan bioasei. Kajian awal bioaktiviti ekstrak mentah serta fraksi daripada kedua- dua spesies Pereskia, seperti kesan antioksidan, antimikrob dan kesitotosikan telah dikaji untuk mengenalpasti ekstrak bioaktif bagi kedua-dua spesies tersebut.

Penyaringan aktiviti kesitotoksikan telah dijalankan menggunakan asei kesitotoksikan in vitro ke atas lima titisan sel karsinoma manusia, iaitu titisan sel karsinoma epidermoid nasofarinks (KB), titisan sel karsinoma serviks (CasKi), titisan sel karsinoma kolon (HCT 116), titisan sel karsinoma payu dara yang melibatkan hormon (MCF7), titisan sel karsinoma paru-paru (A549) serta titisan sel manusia bukan karsinoma (MRC-5).

Ekstrak heksana dan etil asetat bagi kedua-dua spesies Pereskia secara umumnya menunjukkan aktiviti antioksidan yang lebih kuat daripada ekstrak lain.

Ekstrak etil asetat bagi kedua-dua spesies Pereskia juga menunjukkan aktiviti antimikrob yang lemah pada bakteria yang dikaji. Bagi asei kesitotoksikan, ekstrak kedua-dua spesies Pereskia tidak aktif ke atas sel normal MRC-5. Ekstrak etil asetat kedua-dua spesies Pereskia secara umumnya menunjukkan nilai rintangan dan stimulasi yang lebih tinggi ke atas pelbagai titisan sel kanser berbanding dengan ekstrak

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vi lain. Kematian kanser sel yang dirangsang oleh ekstrak aktif kedua-dua spesies Pereskia telah didapati mengaruh kematian sel secara apoptosis berdasarkan tanda fragmentasi DNA yang jelas. Fragmentasi DNA merupakan ciri utama apoptosis.

Tambahan lagi, analisis LUX RT-qPCR [real-time reverse transcriptase–polymerase chain reaction (RT-qPCR) using LUX (Light Upon eXtension) primers] menunjukkan apoptosis yang dirangsang oleh ekstrak aktif ke atas sel kanser tertentu adalah menerusi pengaktifan p53, caspase-3 dan c-myc pada tahap ekspresi yang berbeza.

Metil palmitat, metil linoleat, metil α-linolenat dan fitol telah dikenalpasti daripada ekstrak heksana Pereskia bleo melalui analisis GCMS manakala metil palmitat, metil linoleat, metil α-linolenat dan metil stearat telah dikenalpasti daripada ekstrak heksana Pereskia grandifolia. Daripada hasil penyaringan biologi, ekstrak etil asetat secara umumnya didapati mempunyai aktiviti biologi yang lebih kuat daripada ekstrak yang lain. Kajian kimia seterusnya ditumpukan kepada ekstrak etil asetat bagi kedua-dua spesies Pereskia.

2,4-Di-tert-butilfenol (1), α-tokoferol (2), fitol (3), ß-sitosterol (4), dihidroaktinidiolid (5) dan satu campuran sterol yang mengandungi kampesterol (6), stigmasterol (7) and β-sitosterol (4) telah dipisahkan dan dikenalpasti daripada Pereskia bleo sementara 2,4-di-tert-butilfenol (1), α-tokoferol (2), ß-sitosterol (4) dan satu campuran sebatian yang mengandungi metil palmitat (9), metil oleate (10), metil stearat (11) dan 2,4-di-tert-butilfenol (1) telah dipisahkan dan dikenalpasti daripada Pereskia grandifolia. Perlu diambil perhatian bahawa 2,4-di-tert-butilfenol (1), α-tokoferol (2) dan ß-sitosterol (4) wujud dalam ekstrak etil asetat kedua-dua spesies Pereskia. Kajian aktiviti kesitotosikan sebatian ke atas titisan sel kanser dan penyelidikan selanjutnya ke atas mod tindakan, mencadangkan aktiviti sitotosik sebatian adalah berkaitan dengan induksi apoptosis.

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vii Di samping itu, ketoksikan kedua-dua spesies Pereskia telah dikaji secara in vivo dan hasil eksperimen ketoksikan akut yang dijalankan ke atas tikus menunjukkan ekstrak tumbuhan adalah selamat. Penyaringan untuk Pereskia bleo dan Pereskia grandifolia tempatan menunjukkan kehadiran sejumlah kecil alkaloid sahaja.

Hasil kajian terhadap Pereskia bleo dan Pereskia grandifolia ini memberi fakta saintifik yang sah dalam penggunaan daun kedua-dua Pereskia spesies untuk pengubatan kanser. Kajian mutagenik dan kesan ketoksikan pada organ penting untuk jangka yang lebih panjang perlu dijalankan untuk memastikan tumbuhan tersebut adalah selamat bagi penggunaan manusia.

.

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viii ACKNOWLEDGEMENTS

I wish to express my sincere appreciation and gratitude to my supervisors, Datin Professor Dr. Sri Nurestri Abdul Malek from Institute of Biological Sciences, Faculty of Science and Datin Professor Dr. Norhanom Abdul Wahab from Centre for Foundation Studies in Science, for their continuous supervisions, advices, guidance and continuous encouragements throughout my project. I thank them especially for their patience and the time spent in discussing various approaches and results.

I would also like to express my acknowledgment and appreciation to Dato’

Professor Dr. Hashim Yaacob from International University College of Nursing for the selection of the plant species and his invaluable advices in publishing the findings.

My utmost appreciation to Associate Professor Dr. Kim Kah Hui from Department of Physiology, Faculty of Medicine for making available the facilities in his laboratory for the animal study. Special thanks to Mr V.T. Johgalingam for his willingness in teaching me basic toxicity research techniques.

My appreciation also goes to Associate Professor Koshy Philip from Institute of Biological Sciences, Faculty of Science for the use of laboratory facilities to carry out the antimicrobial screenings. I would also like to thank Mr Saravana Kumar for his guidance in antimicrobial screening work.

I would like to gratefully acknowledge Madam Hong Sok Lai and Mr Lee Guan Serm for their guidance and advice in chemical investigations. My acknowledgement is

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ix also extended to Miss Syarifah Nur Syed Abdul Rahman and Miss Gowri Kanagasabapathy for their help, patience and motivation in carrying out the laboratory experiments.

My sincere appreciation to Miss Sujata Ramasamy for her assistance and generous encouragements in cell culture work. My gratitude towards Miss Wong Kah Hui, Miss Fathiah Abdullah, Miss Maizatul Azmah, Mr Mohd Azrul Mohd Sari, Miss Chan Pek Yue, Miss Lai Li Kuan, Miss Law Ing Kin and Miss Veronica Alicia Yap for their moral support and friendship in providing a good working environment.

My heartfelt gratitude to Mr Yong Yean Kong, Miss Tan Hong Yien, Miss Ong Lai Yee, Miss Teh Siew Phooi, Miss Lee Yeat Mei, Mr Dino Tan Bee Aik and Dr. Andrew Lim for all the help, guidance and patience in the gene expression work, and especially access to the facilities in the laboratory. Special thanks to Mr Yong Yean Kong for sharing all the knowledge in molecular genetics. He has been particularly helpful with suggestions on molecular studies for this work and has never been short of useful comments, ideas and solutions when approached with difficulties.

In addition, I am also very grateful to the staff of the Faculty of Science, Mr Ghana, Puan Latipah, Mr Asokan, Mr Teo, Pak Din, Encik Rufli and Encik Khamis for their support in carrying out the project.

Special thanks to Miss Chaw Sook Ting, Miss Pauline Ong, Miss Stephanie Lau Chiau Hwee and Miss Lim Siew Hua for invaluable moral support, encouragement and many useful comments on my work.

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x Last but not least, my deepest appreciation and love to my husband, Dr Lai Kim Leng for his understanding and endless patience, as well as my parents, sisters and my family-in-law for their encouragements and support which has inspired me to accomplish this study.

I would like to acknowledge the Ministry of Higher Education (MOHE) and University of Malaya for the Skim Latihan Akademik IPTA (SLAI) award that kept me financially sound throughout the study period. This research project was supported by research funds from University of Malaya (PJP F0155/2005D, PPP PS056/2007C, PS241/2008C) and MOHE (FRGS FP040/2008C).

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xi TABLE OF CONTENTS

Page

ABSTRACT ii

ABSTRAK v

ACKNOWLEDGEMENTS viii

TABLE OF CONTENTS xi

LIST OF TABLES xvii

LIST OF FIGURES xx

LIST OF APPENDICES xxv

LIST OF ABBREVIATIONS xxvii

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW 9

2.1 Natural products 9

2.2 Cancer 9

2.2.1 Carcinogens 10

2.2.2 Cell cycle 11

2.2.3 Carcinogenesis 12

2.3 Apoptosis in cancer 13

2.4 Natural products and defence against carcinogenesis 19 2.5 Natural products with conventional therapeutic modalities 20

2.6 The Cactaceae family 21

2.7 The Pereskia genus 22

2.7.1 The Pereskia bleo 24

2.7.2 The Pereskia grandifolia 28

2.8 Bioactivity assays 32

2.9 Antioxidant activity 33

2.9.1 Oxygen and singlet oxygen 33

2.9.2 Free radicals and Reactive Oxygen Species (ROS) 33 2.9.3 Cellular effects of oxidative stress and related diseases 34 2.9.4 Biological antioxidant defence mechanisms 35

2.9.5 Synthetic and natural antioxidants 36

2.9.6 Methods to determine antioxidant activities 38

2.10 Antimicrobial activity 39

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xii

2.11 In vitro cytotoxic activity tests 42

2.11.1 In vitro testing system 42

2.11.2 In vitro cytotoxic activity testing 44 2.11.3 Methods to determine cytotoxic activity 45

2.12 Apoptosis screening and detection 45

2.12.1 Detection of morphological changes 46

2.12.2 Detection of DNA fragmentation 47

2.12.3 TUNEL assay 48

2.13 Determination of expression level of apoptotic-related genes 49

2.13.1 Introduction to gene expression 49

2.13.2 Importance of gene expression measurement 50

2.13.3 Apoptotic-related genes 51

2.13.4 Methods of mRNA quantification 54

2.13.5 RT-PCR quantification 55

(i) Conventional RT-PCR quantification 56

(ii) The RT-qPCR quantification 57

2.13.6 Fluorogenic LUX primer RT-qPCR assay 60

2.14 Acute oral toxicity assessment 64

3 MATERIALS AND METHODS 67

3.1 Plant materials 67

3.2 Extraction and fractionation of methanol extract of plant samples 67

3.3 Antioxidant activity 68

3.3.1 Folin-Ciocalteu method 68

3.3.2 Scavenging activity on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals

70

3.3.3 Reducing power assay 73

3.3.4 ß-Carotene bleaching method 73

3.3.5 Statistical analysis 74

3.4 Antimicrobial activity 75

3.4.1 Preparation of agar and broth 75

3.4.2 Test microorganisms and microbial culture 75

3.4.3 Agar disc diffusion assay 76

3.4.4 Broth dilution assay 78

3.4.5 Statistical analysis 79

3.5 In vitro cytotoxicity assay 79

3.5.1 Cell lines and culture medium 79

3.5.2 In vitro neutral red cytotoxicity assay 80

3.5.3 Statistical analysis 82

3.6 Detection of DNA fragmentation (apoptosis) 82

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xiii 3.7 Determination of the expression level of apoptotic-related genes 83

3.7.1 Preparation of quantitative standard (QSTD) for real-time quantification

84 3.7.2 Validation and optimization of qPCR assay using LUX

primers

91 3.7.3 Application of optimized qPCR assay to experiment set 99

3.7.4 Statistical analysis 102

3.8 Acute oral toxicity 104

3.8.1 Test species 104

3.8.2 Procedure of acute oral toxicity 105

3.9 Extraction, isolation and identification of chemical constituents from the bioactive extracts

106

3.9.1 Instrumentation 106

3.9.2 Column chromatography 107

3.9.3 Thin Layer Chromatography (TLC) 107

3.9.4 Preparative-Thin Layer Chromatography (Prep-TLC) 108 3.9.5 Identification of compounds in hexane extracts of P. bleo

and P. grandifolia using GCMS

108 3.9.6 Extraction and isolation of chemical constituents from the

bioactive ethyl acetate extract of P. bleo

109 3.9.7 Extraction and isolation of chemical constituents from the

bioactive ethyl acetate extract of P. grandifolia

114

3.10 Screening for alkaloids 118

3.10.1 Preparation of Wagner’s reagent 118

3.10.2 Preliminary alkaloidal test 118

3.10.3 Confirmatory test for alkaloids 119

3.10.4 Test for quaternary and/or amine oxide bases 119

4 RESULTS AND DISCUSSIONS 120

4.1 Extraction yield of P. bleo and P. grandifolia 120 4.2 Antioxidant activity of P. bleo and P. grandifolia extracts 122

4.2.1 Determination of reducing capacity of P. bleo and P.

grandifolia extracts using Folin-Ciocalteau method

122 4.2.2 Scavenging activity of P. bleo and P. grandifolia extracts

on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals

126 4.2.3 Reducing power assay of P. bleo and P. grandifolia

extracts

132 4.2.4 ß-Carotene bleaching activity of P. bleo and P.

grandifolia extracts

137 4.2.5 Comparison of antioxidant activity of P. bleo and P.

grandifolia extracts

141

4.3 Antimicrobial activity of P. bleo and P. grandifolia extracts 142 4.3.1 Agar disc diffusion assay of P. bleo and P. grandifolia

extracts

144

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xiv

4.3.2 Broth dilution assay 147

(i) MIC and MBC of P. bleo extracts 147 (ii) MIC and MBC of P. grandifolia extracts 148 4.3.3 Comparison of antimicrobial activity of P. bleo and P.

grandifolia

150

4.4 In vitro neutral red cytotoxicity assay 151

4.4.1 Cytotoxic activity of P. bleo and P. grandifolia extracts 154 (i) Human nasopharyngeal epidermoid carcinoma cell

line (KB)

154 (ii) Human cervical carcinoma cell line (CasKi) 156 (iii) Human colon carcinoma cell line (HCT 116) 158 (iv) Hormone-dependent breast carcinoma cell line

(MCF7)

160

(v) Lung carcinoma cell line (A549) 162

(vi) Human fibroblast cell line (MRC-5) 164

4.4.2 Cytotoxic activity of doxorubicin 166

4.4.3 Comparison of cytotoxic activity of P. bleo and P.

grandifolia

168

4.5 Detection of DNA fragmentation 170

4.5.1 DeadEndTM Colorimetric Apoptosis Detection System (Promega)

171 4.5.2 Induction of apoptosis by the cytotoxic active extracts of

P. bleo and P. grandifolia on the selected human cancer cells

172

4.6 Determination of the expression level of apoptotic-related genes 173 4.6.1 The choice of using LUX RT-qPCR assay in gene

expression study

175 4.6.2 Validation and optimization of the LUX RT-qPCR assay 176

(i) Specificity test of LUX primers 177

(ii) Optimization of the concentrations of LUX primers and MgCl2

182 (iii) Optimization of the annealing temperature (TA) 182 (iv) Determination of the lowest detection limit and

sensitivity of the LUX RT-qPCR assay

183 4.6.3 Considerations for RT-qPCR procedures 185

(i) Reagents 185

(ii) Negative controls 185

(iii) Internal reference gene 186

4.6.4 Data analysis 187

4.6.5 Expression level of apoptosis-related genes in the cytotoxic active extract-treated cells

189 (i) Expression level of apoptosis-related genes in the

P. bleo cytotoxic active extract-treated cells

189 (ii) Expression level of apoptosis-related genes in the

P. grandifolia cytotoxic active extract-treated cells

193 (iii) Summary of expression level of apoptosis-related

genes in the Pereskia spp. cytotoxic active extract-

196

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xv treated cells

4.7 Acute oral toxicity assessment of P. bleo and P. grandifolia crude extracts

197

4.8 Chemical constituents from the bioactive extracts of P. bleo and P.

grandifolia

199 4.8.1 Identification of compounds in hexane extract of P. bleo

using GCMS

200 4.8.2 Identification of compounds in hexane extract of P.

grandifolia using GCMS

204 4.8.3 Chemical constituents from the bioactive ethyl acetate

extract of P. bleo

207 (i) Structural determination of 2,4-ditert-butylphenol

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209 (ii) Structural determination of α-tocopherol (2) 212 (iii) Structural determination of phytol (3) 214 (iv) Structural determination of ß-sitosterol (4) 215 (v) Structural determination of dihydroactinidiolide (5) 217 (vi) Structural determination of mixture of sterols

(mixture A)

218 4.8.4 Chemical constituents from the bioactive ethyl acetate

extract of P. grandifolia

221 (i) Structural determination of 2,4-ditert-butylphenol

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223 (ii) Structural determination of α-tocopherol (2) 224 (iii) Structural determination of ß-sitosterol (4) 225 (iv) Structural determination of phytone (8) 226 (v) Structural determination of mixture B 227 4.9 Cytotoxic and apoptosis effects of chemical constituents isolated

from the bioactive ethyl acetate extracts of P. bleo and P.

grandifolia

229 4.9.1 Cytotoxic and apoptosis effects of 2,4-di-tert-butylphenol

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230 (i) Cytotoxic activity of 2,4-di-tert-butylphenol (1) 230 (ii) Induction of apoptosis by 2,4-di-tert-butylphenol

(1) on selected cells

232 (iii) Expression level of apoptosis-related genes in 2,4-

di-tert-butylphenol (1)-treated cells

232 4.9.2 Cytotoxic and apoptosis effects of α-tocopherol (2) 237 (i) Cytotoxicity activity of α-tocopherol (2) 237 (ii) Induction of apoptosis by α-tocopherol (2) on

selected cells

238 (iii) Expression level of apoptosis-related genes in α-

tocopherol (2)-treated cells

239

4.9.3 Cytotoxic effect of phytol (3) 241

4.9.4 Cytotoxic effect of ß-sitosterol (4) 243 4.9.5 Cytotoxic and apoptosis effects of dihydroactinidiolide (5) 245 (i) Cytotoxic activity of dihydroactinidiolide (5) 245

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xvi (ii) Induction of apoptosis by dihydroactinidiolide (5)

on HCT 116 cells

246 (iii) Expression level of apoptosis-related genes in

dihydroactinidiolide (5)-treated HCT 116 cells

246 4.9.6 Cytotoxic effect of mixture of sterols (mixture A) 247 4.9.7 Cytotoxic and apoptosis effects of phytone (8) 249 (i) Cytotoxic activity of phytone (8) 249 (ii) Induction of apoptosis by phytone (8) on selected

cells

250 (iii) Expression level of apoptosis-related genes in

phytone (8)-treated cells

250 4.9.8 Cytotoxic and apoptosis effects of mixture B 253 (i) Cytotoxic activity of mixture B 253 (ii) Induction of apoptosis by mixture B on selected

cells

255 (iii) Expression level of apoptosis-related genes in

mixture B-treated cells

255 4.9.9 Comparison of cytotoxic and apoptosis effects of

chemical constituents isolated from the bioactive extracts of P. bleo and P. grandifolia

257

4.10 Screening for alkaloids of P. bleo and P. grandifolia 261

5 CONCLUSION 263

REFERENCES 270

APPENDICES 297

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xvii LIST OF TABLES

Table Page

2.1 Endpoints used to identify cytotoxic effects using in vitro test system

43

2.2 Typical methods to study different aspect of apoptosis 46

2.3 GHS acute oral toxicity classifications 66

3.1 The preparation of different concentrations of gallic acid solution for calibration plot

69

3.2 Preparation of reaction mixture of selected extracts or fractions, DPPH and methanol

71

3.3 Preparation of reaction mixture of selected extracts, DPPH and methanol

72 3.4 Reaction mixture of positive reference standards, DPPH and

methanol for DPPH assay

72

3.5 Reaction components for one-step RT-PCR 87

3.6 Thermal cycling profile of one-step RT-PCR 88

3.7 The sequence of primers used in RT-PCR for preparation of QSTD 89

3.8 The sequence of LUX primers used in qPCR 93

3.9 Reaction components for qPCR 94

3.10 Thermal cycling and melting profile of qPCR 95

3.11 DNA-primer mix preparation for optimization assay 96

3.12 Reaction components for qPCR 97

3.13 Concentrations of Primer-MgCl2 mix for optimization assay 98

3.14 Reaction components for qPCR 98

3.15 Reaction components for cDNA synthesis 101

4.1 Yield of methanol extracts of P. bleo and P. grandifolia 120 4.2 Yield of extracts fractionated from P. bleo crude methanol extract 121

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xviii 4.3 Yield of extracts fractionated from P. grandifolia crude methanol

extract

121

4.4 Reducing capacity of P. bleo and P. grandifolia extracts 124 4.5 The scavenging activity (IC50 values) of P. bleo and P. grandifolia

extracts on the inhibition of scavenging activity of DPPH radicals

129 4.6 Reducing powers of extracts of P. bleo and P. grandifolia at various

concentrations

135

4.7 Antioxidant activity (%) of P. bleo and P. grandifolia extracts measured by ß-carotene bleaching method

139 4.8 Results of the antimicrobial tests of the investigated plants in agar

diffusion assay

146

4.9 MIC and MBC of P. bleo and P. grandifolia extracts after 24 h incubation with bacteria

149

4.10 The IC50 values of P. bleo and P. grandifolia extracts tested against KB cell line

156 4.11 The IC50 values of P. bleo and P. grandifolia extracts tested against

CasKi cell line

158

4.12 The IC50 values of P. bleo and P. grandifolia extracts tested against HCT 116 cell line

160 4.13 The IC50 values of P. bleo and P. grandifolia extracts tested against

MCF7 cell line

162

4.14 The IC50 values of P. bleo and P. grandifolia extracts tested against A549 cell line

164

4.15 The IC50 values of P. bleo and P. grandifolia extracts tested against MRC-5 cell line

166 4.16 The IC50 values of doxorubicin against various cancer and non-

cancer cell lines tested

167

4.17 Comparison between IC50 values of P. bleo and P. grandifolia extracts against various cancer and non-cancer cell lines

170 4.18 Comparison of chemistry options for real-time amplification 176 4.19 Results of the potential toxic effect of the crude extracts of P. bleo

and P. grandifolia in mice

199

4.20 Cytotoxic activity (IC50 values) of 2,4-di-tert-butylphenol (1) against 231

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xix selected human cell lines

4.21 Cytotoxic activity (IC50 values) of α-tocopherol (2) against selected human cell lines

238

4.22 Cytotoxic activity (IC50 values) of phytol (3) against selected human cell lines

243 4.23 Cytotoxic activity (IC50 values) of ß-sitosterol (4) against selected

human cell lines

244

4.24 Cytotoxic activity (IC50 values) of dihydroactinidiolide (5) against selected human cell lines

246 4.25 Cytotoxic activity (IC50 values) of mixture of sterols (mixture A)

against selected human cell lines

248

4.26 Cytotoxic activity (IC50 values) of phytone (8) against selected human cell lines

250

4.27 Cytotoxic activity (IC50 values) of mixture B against selected human cell lines

255 4.28 Cytotoxic activity (IC50 values) of chemical constituents isolated

from the bioactive ethyl acetate extracts of P. bleo and P.

grandifolia

260

4.29 Cytotoxic activity (IC50 values) of bioactive extracts and chemical constituents isolated from the bioactive ethyl acetate extract of P.

bleo

261

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xx LIST OF FIGURES

Figures Page

1.1 Outline of general procedures 7

2.1 The cell cycle 12

2.2 Main steps in apoptosis 14

2.3 The two main routes for triggering apoptosis 16

2.4 Mechanism of apoptosis 18

2.5 The appearance of P. bleo in Seksyen 17, Petaling Jaya, Selangor, Malaysia

25

2.6 The stem of P. bleo 25

2.7 The hemispherical fruit of P. bleo 26

2.8 The orangish-red flowers of P. bleo 26

2.9 The appearance of P. grandifolia in Seksyen 17, Petaling Jaya, Selangor, Malaysia

29

2.10 The stem of P. grandifolia 30

2.11 The pink flowers of P. grandifolia 30

2.12 Alkaloids isolated from P. bleo and P. grandifolia 31 2.13 Compounds isolated from dried powdered fruits of P. grandifolia 32

2.14 Steps in gene expression process 50

2.15 General PCR schema 56

2.16 RT-PCR quantification 57

2.17 The qPCR amplification curve 59

2.18 LUX primer reaction 62

2.19 Melting curve analysis confirming specific amplification with LUX primers

64 3.1 Example of the results of the agar disk diffusion assay 77

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xxi 3.2 Induction of apoptosis by the DNase I and DMSO on the KB cells 83

3.3 Example of result analyzed by the software 104

3.4 The extraction and fractionation procedures leading to the isolation of chemical constituents from the bioactive ethyl acetate extract of P. bleo

112

3.5 The extraction and isolation of compounds 1-5 and mixture A from the bioactive ethyl acetate extract of P. bleo

113

3.6 The extraction and fractionation procedures leading to the isolation of chemical constituents from the bioactive ethyl acetate extract of P. grandifolia

116

3.7 The extraction and isolation of compounds 1, 2, 4, 8 and mixture B from the bioactive ethyl acetate extract of P. grandifolia

117

4.1 The gallic acid calibration graph 124

4.2 Reducing capacity of P. bleo and P. grandifolia extracts 125 4.3 The principal of DPPH radical scavenging assay 127 4.4 Scavenging effect of P. bleo extracts on DPPH radical 128 4.5 Scavenging effect of P. grandifolia extracts on DPPH radical 128 4.6 Scavenging effect of positive reference standards (BHA and

ascorbic acid) on DPPH radical

129

4.7 Scavenging effect of lower concentrations of P. bleo extracts on DPPH radical to determine the IC50 values

130 4.8 Scavenging effect of lower concentrations of P. grandifolia extracts

on DPPH radical to determine the IC50 values

131

4.9 Reducing powers of ascorbic acid and BHA (reference compounds) 136 4.10 Reducing powers of extracts of P. bleo at various concentrations 136 4.11 Reducing powers of extracts of P. grandifolia at various

concentrations

137 4.12 Antioxidant activity (%) of P. bleo extracts measured by ß-carotene

bleaching method

140

4.13 Antioxidant activity (%) of P. grandifolia extracts measured by ß- carotene bleaching method

140

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xxii 4.14 The in vitro growth inhibitions of KB cells by P. bleo extracts

determined by neutral red cytotoxicity assay

155

4.15 The in vitro growth inhibitions of KB cells by P. grandifolia extracts determined by neutral red cytotoxicity assay

155 4.16 The in vitro growth inhibitions of CasKi cells by P. bleo extracts

determined by neutral red cytotoxicity assay

157

4.17 The in vitro growth inhibitions of CasKi cells by P. grandifolia extracts determined by neutral red cytotoxicity assay

157 4.18 The in vitro growth inhibitions of HCT 116 cells by P. bleo

extracts determined by neutral red cytotoxicity assay

159

4.19 The in vitro growth inhibitions of HCT 116 cells by P. grandifolia extracts determined by neutral red cytotoxicity assay

159

4.20 The in vitro growth inhibitions of MCF7 cells by P. bleo extracts determined by neutral red cytotoxicity assay

161 4.21 The in vitro growth inhibitions of MCF7 cells by P. grandifolia

extracts determined by using neutral red cytotoxicity assay

161

4.22 The in vitro growth inhibitions of A549 cells by P. bleo extracts determined by neutral red cytotoxicity assay

163

4.23 The in vitro growth inhibitions of A549 cells by P. grandifolia extracts determined by neutral red cytotoxicity assay

163 4.24 The in vitro growth inhibitions of MRC-5 cells by P. bleo extracts

determined by neutral red cytotoxicity assay

165

4.25 The in vitro growth inhibitions of MRC-5 cells by P. grandifolia extracts determined by neutral red cytotoxicity assay

165 4.26 The in vitro growth inhibitions of selected human cells by

doxorubicin as positive reference standard determined by neutral red cytotoxicity assay

167

4.27 The specificity of FAM-labelled LUX primers in green channel 179 4.28 The specificity of JOE-labelled LUX primers in yellow channel 180 4.29 Melting curve of FAM-labelled LUX primers in green channel 180 4.30 Melting curve of JOE-labelled LUX primers in yellow channel 181 4.31 The standard curve and amplification curve obtained by the LUX

qPCR assays on the dilution series of ß-actin QSTD template

184

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xxiii 4.32 The mRNA expression of p53, caspase-3 and c-myc detected in KB

cells treated with methanol extract of P. bleo for different durations 191

4.33 The mRNA expression of p53, caspase-3 and c-myc detected in KB cells treated with ethyl acetate extract of P. bleo for different durations

192

4.34 The mRNA expression of p53, caspase-3 and c-myc detected in KB cells treated with hexane extract of P. grandifolia for different durations

194

4.35 The mRNA expression of p53, caspase-3 and c-myc detected in KB cells treated with ethyl acetate extract of P. grandifolia for different durations

195

4.36 The mRNA expression of p53, caspase-3 and c-myc detected in MCF 7 cells treated with ethyl acetate extract of P. grandifolia for different durations

196

4.37 Compounds identified from the hexane extract of P. bleo using GCMS analysis

201

4.38 Compounds identified from the hexane extract of P. grandifolia using GCMS analysis

204 4.39 McLafferty rearrangement of methyl palmitate 207 4.40 Chemical constituents from the bioactive ethyl acetate extract of P.

bleo

208

4.41 Chemical constituents from the bioactive ethyl acetate extract of P.

grandifolia

222 4.42 The in vitro growth inhibitions of 2,4-di-tert-butylphenol (1)

against selected human cell lines determined by neutral red cytotoxicity assay

231

4.43 The mRNA expression of p53, caspase-3 and c-myc detected in KB cells treated with 2,4-di-tert-butylphenol (1) for different durations

233

4.44 The mRNA expression of p53, caspase-3 and c-myc detected in MCF7 cells treated with 2,4-di-tert-butylphenol (1) for different durations

234

4.45 The mRNA expression of p53, caspase-3 and c-myc detected in CasKi cells treated with 2,4-di-tert-butylphenol (1) for different durations

235

4.46 The mRNA expression of p53, caspase-3 and c-myc detected in A549 cells treated with 2,4-di-tert-butylphenol (1) for different durations

236

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xxiv 4.47 The in vitro growth inhibitions of α-tocopherol (2) against selected

human cell lines determined by neutral red cytotoxicity assay

238

4.48 The mRNA expression of p53, caspase-3 and c-myc detected in CasKi cells treated with α-tocopherol (2) for different durations

240 4.49 The mRNA expression of p53, caspase-3 and c-myc detected in

A549 cells treated with α-tocopherol (2) for different durations

241

4.50 The in vitro growth inhibitions of phytol (3) against selected human cell lines determined by neutral red cytotoxicity assay

242 4.51 The in vitro growth inhibitions of ß-sitosterol (4) against selected

human cell lines determined by neutral red cytotoxicity assay

244

4.52 The in vitro growth inhibitions of dihydroactinidiolide (5) against selected human cell lines determined by neutral red cytotoxicity assay

245

4.53 The mRNA expression of p53, caspase-3 and c-myc detected in HCT 116 cells treated with dihydroactinidiolide (5) for different durations

247

4.54 The in vitro growth inhibitions of mixture of sterols (mixture A) against selected human cell lines determined by neutral red cytotoxicity assay

248

4.55 The in vitro growth inhibitions of phytone (8) against selected human cell lines determined by neutral red cytotoxicity assay

249

4.56 The mRNA expression of p53, caspase-3 and c-myc detected in CasKi cells treated with phytone (8) for different durations

251 4.57 The mRNA expression of caspase-3 and c-myc detected in CasKi

cells treated with phytone (8) for different durations

252

4.58 The mRNA expression of p53, caspase-3 and c-myc detected in HCT 116 cells treated with phytone (8) for different durations

253 4.59 The in vitro growth inhibitions of mixture B against selected

human cell lines determined by neutral red cytotoxicity assay

254

4.60 The mRNA expression of p53, caspase-3 and c-myc detected in CasKi cells cells treated with mixture B for different durations

256

4.61 The mRNA expression of p53, caspase-3 and c-myc detected in A549 cells treated with mixture B for different durations

257

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xxv LIST OF APPENDICES

Appendices Page

A Cell culture techniques 297

B Gas chromatogram of hexane extract of P. bleo 301

B1 Mass spectrum of methyl palmitate 301

B2 Mass spectrum of methyl linoleate 302

B3 Mass spectrum of methyl α-linolenate 302

B4 Mass spectrum of phytol 303

C Gas chromatogram of hexane extract of P. grandifolia 304

C1 Mass spectrum of methyl palmitate 304

C2 Mass spectrum of methyl linoleate 305

C3 Mass spectrum of methyl α-linoleate 305

C4 Mass spectrum of methyl stearate 306

D Mass spectrum of 2,4-di-tert-butylphenol (1) 306

D1 1H-NMR spectrum of 2,4-di-tert-butylphenol (1) 307 D2 13C-NMR spectrum of 2,4-di-tert-butylphenol (1) 308

D3 DEPT spectrum of 2,4-di-tert-butylphenol (1) 309

E Mass spectrum of α-tocopherol (2) 310

F Mass spectrum of phytol (3) 310

F1 1H-NMR spectrum of phytol (3) 311

F2 13C-NMR spectrum of phytol (3) 312

F3 DEPT spectrum of phytol (3) 313

G Mass spectrum of ß-sitosterol (4) 314

G1 1H-NMR spectrum of ß-sitosterol (4) 315

G2 13C-NMR spectrum o f ß-sitosterol (4) 316

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xxvi

H Mass spectrum of dihydroactinidiolide (5) 317

I Gas chromatogram of mixture A 317

I1 Mass spectrum of campesterol (6) 318

I2 Mass spectrum of stigmasterol (7) 318

I3 Mass spectrum of ß-sitosterol (4) 319

J Mass spectrum of 2,4-di-tert-butylphenol (1) 319

K Mass spectrum of α-tocopherol (2) 320

L Mass spectrum of ß-sitosterol (4) 320

M Mass spectrum of phytone (8) 321

N Gas chromatogram of mixture B 321

N1 Mass spectrum of 2,4-di-tert-butylphenol (1) 322

N2 Mass spectrum of methyl palmitate (9) 322

N3 Mass spectrum of methyl oleate (10) 323

N4 Mass spectrum of methyl stearate (11) 323

O List of publications 324

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xxvii LIST OF ABBREVIATIONS

ANOVA ANalysis Of VAriance

ATCC American Tissue Culture Collection A549 Human lung carcinoma cell line BHA

BLAST CasKi CC CDCl3

cDNA

Butylated hydroxyanisole

Basic Local Alignment and Search Tool Human cervical carcinoma cell line Column chromatography

Deuterated chloroform

Complementary deoxyribonucleic acid CH2Cl2 Dichloromethane

CHCl3 Chloroform CO2

Ct

°C DEPC DEPT

Carbon dioxide Threshold cycle Degree Celsius

Diethyl pyrocarbonate

Distortionless Enhancement by Polarisation Transfer DMSO

DNA dNTP

Dimethyl sulfoxide Deoxyribonucleic acid

Deoxyribonucleic triphosphate DPPH

ds

1,1-Diphenyl-2-picrylhydrazyl Double stranded

EDTA Ethylene diamine tetra acetic acid ELISA Enzyme-linked immunosorbent assay EtOAc Ethyl acetate

FAM 6-carboxy-fluorescein FBS Foetal Bovine Serum

g Gram

GCMS Gas Chromatography Mass Spectroscopy HCT 116 Human colon carcinoma cell line

HEPES HPLC h

N-2-Hydroxylethyl-Piperazine-N-2-Ethane-Sulfonoc High Pressured Liquid Chromatography

Hour

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xxviii

H2O Water

IC50

IR

Inhibition Concentration at 50 % Infra Red

JOE KB kg

6-carboxy-4’,5’-dichloro-2’,7’-dimethoxy-flurescein Human nasopharyngeal epidermoid carcinoma cell line Kilogram

L LD50

LUX

Litre

Lethal Dose at 50 % Light Upon eXtension

µg Microgram

µl Microlitre

µm Micrometer

MBC Minimum Bacterial Concentration

MCF7 Hormone-dependent breast carcinoma cell line

MeOH Methanol

Mg MgCl2

Milligram

Magnesium chloride

MIC Minimum Inhibitory Concentration

min Minute

ml Millilitre

Mm MRC-5 mRNA MS

Millimetre

Non-cancer human fibroblast cell line Messenger RNA

Mass Spectroscopy NaCl Sodium chloride Na2CO3 Sodium carbonate NaHCO3

NMR

Sodium bicarbonate

Nuclear Magnetic Resonance

nm Nanometre

NRTC Non-reverse-transcriptase control NTC Non-template control

OD Optical Density

PBS PCR

Phosphate buffered saline Polymerase Chain Reaction

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xxix Prep-TLC

qPCR QSTD RNA ROS

Preparative-Thin Layer Chromatography Real-time polymerase chain reaction Quantitative standard

Ribonucleic acid

Reactive oxygen species rpm

RT-PCR RT-qPCR s

SD

Rotation per minute

Reverse-transcriptase polymerase chain reaction

Real-time reverse-transcriptase polymerase chain reaction Second

Standard deviation spp.

ss TA

TBE buffer Tm

Taq TLC TUNEL

Species

Single stranded

Annealing temperature Tris-Borate-EDTA buffer Melting temperature Thermus aquaticu

Thin Layer Chromatography

Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling

UV Ultraviolet

% Percentage

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1 CHAPTER 1

INTRODUCTION

Today, naturally derived products play an important role as source of medicine.

Many pharmaceutical agents have been discovered by screening natural products from plants and marine organisms. The structural diversity of compounds derived from natural products provides valuable sources of novel lead compounds against newly discovered therapeutic targets (Harvey, 1999).

The statistics regarding the uses of natural products as drug are now well-known and have been repeatedly presented and discussed. Over the period 1981-2002, 1031 new chemical entities (NCEs) have been discovered. Thus, in the area of cancer the percentage of new chemical entities that are non-synthetic has remained at 62 % averaged over the whole time frame. In the antihypertensive area, of the 74 formally known synthetic drugs, 48 can be traced to natural product structures/ mimics (Newman et al., 2003; Abas, 2005).

There are around 250,000 plant species in the world and 60 % of them are located in the tropical rainforests. The plant resources of Malaysia comprise about 15,000 species of higher plants. It was estimated that about 1,000 of these plants have undergone simple chemical screening and much less have been subjected to thorough chemical or pharmacological studies (Goh et al., 1993). The huge diversity of the Malaysian flora means that we can expect well diversed chemical structures from their secondary metabolites, and chemical diversity is one of the plus factors that makes natural products excellent candidates for any screening programme.

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2 Ethnopharmacological data has been one of the common useful ways for the discovery of biological active compounds from plants, in which the selection of a plant is based on the prior information on the folk medicine use of the plant. It is generally known that ethnomedical data provides substantially increased chance of finding active plants relative to random approach (Chapuis et al., 1988; Cordell et al., 1991).

Pereskia bleo (P. bleo) and Pereskia grandifolia (P. grandifolia), commonly known as the ‘Jarum Tujuh Bilah’ in Malay and ‘Cak Sing Cam’ in Chinese belong to the botanical family Cactaceae. P. bleo can be easily mistaken from P. grandifolia because they look similar vegetatively. However, they can be distinguished by the leaves, flowers and spines. P. bleo have thinner and corrugated leaves, orangish-red flowers with shorter spines compared to P. grandifolia. In contrast, P. grandifolia have thicker and uncorrugated leaves, pink to purple-pink flowers with longer and lesser spines.

Both P. bleo and P. grandifolia have been used as natural remedy in cancer- related diseases, either eaten raw or taken as a concoction brewed from fresh plant. The leaves are also taken as vegetables by some natives. Both are believed to have anti- cancer, anti-tumour, anti-rheumatic, anti-ulcer and anti-inflammatory properties. They are also used as remedy for the relief of headache, gastric pain, ulcers, haemorrhoids and atopic dermatitis; and refresh the body (Goh, 2000; Rahmat, 2004; Tan et al., 2005).

The pounded leaf paste of P. bleo is also applied to the wound or cut for pain relief (Kehidupan Sihat, 2006). In Panama, the locals use the whole plant of P. bleo to treat the gastrointestinal problems (Gupta et al., 1996). On the other hand, P. grandifolia is also used to reduce swellings (Sahu et al., 1974; Anon, 1969).

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3 Although P. bleo and P. grandifolia are reported to be used in a large number of Malaysian traditional medicine preparations, there is not much recorded data on biological studies and chemical investigations of P. bleo and P. grandifolia. There is only one phytochemical report (Doetsch et al., 1980) and four biological studies (Matsuse et al., 1999; Tan et al., 2005; Ruegg et al., 2006; Er et al., 2007) reported for P. bleo. Similarly, very little phytochemical work (Doetsch et al., 1980; Sahu et al., 1974) and biological study (Ooi et al., 2003) has been reported for P. grandifolia.

The experimental approach in the present study is based on bioassay-guided fractionation. In this endeavour, the crude methanol and fractionated extracts of P. bleo and P. grandifolia were firstly prepared for the biological assessment. The extracts were subjected to antioxidant assays, antimicrobial assays and neutral red cytotoxicity assay to identify the bioactive extracts of both Pereskia species. The cytotoxic active extracts were further subjected to detection of DNA fragmentation (apoptosis) and determination of the expression level of apoptotic–related genes to verify the possible mechanisms of cell death elicited by the extracts on the cells.

After identifying the bioactive extracts of P. bleo and P. grandifolia, the chemical constituents responsible for the bioactivities were identified. Thus, the bioactive extracts were subjected to isolation and purification procedures to obtain chemical constituents present in the extracts. The isolated chemical constituents were further tested for their cytotoxic activity against the selected human cell lines. The active cytotoxic chemical constituents were then subjected to detection of apoptosis and determination of the expression level of apoptotic–related genes.

Acute oral toxicity was also undertaken in the present study to determine the safety parameters of the leaves of both Pereskia spp. as in vitro trials did not always

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4 reflect the outcome of in vivo studies. In addition, the locally grown P. bleo and P.

grandifolia were screened for alkaloids in the present study to confirm the presence of alkaloids as Doetsch et al. (1980) reported the isolation of alkaloids in both Pereskia spp. The general procedures in the present study are outlined in Figure 1.1.

In the present study, the antioxidant activities of the extracts were determined by four different assays, namely scavenging activity of plant extracts on 1, 1-diphenyl- 2-picrylhydrazyl (DPPH) radicals, reducing power assay, ß-carotene method and Folin- Ciocalteau’s method. To our knowledge, there is no antioxidant study reported for both P. bleo and P. grandifolia.

The antimicrobial activities of the extracts were determined by agar diffusion and broth dilution method. The broth dilution method was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). To our knowledge, there is only one report on the antimicrobial properties of P.

bleo (Ruegg et al., 2006) and no such report on P. grandifolia.

Determination of cytotoxic effect of Pereskia spp. extracts and isolated chemical constituents were investigated on six human cell lines, which were the human nasopharyngeal epidermoid carcinoma cell line (KB), human cervical carcinoma cell line (CasKi), human colon carcinoma cell line (HCT 116), hormone-dependent breast carcinoma cell line (MCF7), human lung carcinoma cell line (A549) and non-cancer human fibroblast cell line (MRC-5). To our knowledge, no report on the cytotoxicity of P. bleo and P. grandifolia against the above mentioned human cancer cell lines has ever been published.

Apoptosis or programmed cell death plays important roles in many biological processes including carcinogenesis, tumorigenesis and cancer. Many chemotherapeutic

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5 and chemopreventive agents have been found to induce apoptotic cell death (Kong et al., 2001). In the present study, DNA fragmentation (apoptosis) was detected using a modified TUNEL [terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling] assay.

Subsequently, the protocol for the evaluation of mRNA expression levels of apoptosis-related genes by LUX RT-qPCR [real-time reverse transcriptase–polymerase chain reaction (RT-qPCR) using LUX (Light Upon eXtension) primers] analysis has been developed in the present study. This LUX RT-qPCR assay offers significant advantages with respect to the rapidity, sensitivity and reproducibility of quantification assay for gene expression.

Objectives of study

The main objectives of the present study were as follows:

a. to screen for antioxidant, antimicrobial and in vitro cytotoxic activities of P.

bleo and P. grandifolia extracts.

b. to investigate the ability of cytotoxic active extracts of P. bleo and P.

grandifolia to induce apoptosis and to determine the possible mechanisms of cell death elicited by the extracts on selected cancer cells.

c. to isolate the chemical constituents from the identified bioactive extracts of P.

bleo and P. grandifolia through the bioassay guided fractionation technique.

d. to identify and elucidate the structures of the chemical constituents by using modern spectroscopic methods.

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6 e. to evaluate the cytotoxic activities of the chemical constituents, their ability to induce apoptosis and possible mechanisms of cell death elicited by the chemical constituents on selected cancer cells.

f. to develop a protocol for the evaluation of mRNA expression levels of apoptosis-related genes by LUX RT-qPCR assay [real-time reverse transcriptase–polymerase chain reaction (RT-qPCR) using LUX (Light Upon eXtension) primers].

g. to determine the safety parameters of P. bleo and P. grandifolia extracts by using acute oral toxicity assessment.

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7

……continued Fresh leaves of Pereskia spp.

Washed, dried and ground Dried and ground plant material

Extraction with methanol (3x)

Concentration under reduced pressure Methanol extract

Extraction with hexane

Hexane-soluble fraction Hexane-insoluble fraction Partition (v/v) Ethyl acetate: water (1:1)

Ethyl acetate fraction Water fraction

Biological screenings:

1. Antioxidant activity 2. Antimicrobial activity 3. Cytotoxic activity

Detection of DNA fragmentation (apoptosis) If cytotoxic active

If active

Determination of the expression level of apoptotic-related genes

Acute oral toxicity Screening for alkaloids

Identified bioactive fractions GCMS

analysis

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8 Figure 1.1, continued

Figure 1.1: Outline of general procedures Identified bioactive fractions

Column chromatography, TLC, prep-TLC and HPLC

Chemical constituents

Spectroscopic and spectrometric identification (NMR, MS) Identified chemical constituents

Screening of cytotoxic activity

If active If active

Determination of the expression level of apoptotic-related genes Detection of DNA fragmentation (apoptosis)

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9 CHAPTER 2

LITERATURE REVIEW

2.1 Natural products

Natural products usually refer to secondary metabolites which has relatively complex structures. Secondary metabolites are usually characteristic of specific botanical sources in comparison to primary metabolites which occur in almost every plant. A general characteristic of natural products is that few of them have a clearly recognized function in the metabolic activities of the organisms in which they are found (Geissman et al., 1969).

Natural products can be classified into various families, such as alkaloids, terpenoids and phenolics including flavonoids. Natural products perform various functions, and many of them have interesting and useful biological activities (Harvey, 1999). The utility of natural products as a source of novel structures is still alive and well. The number of plants used as medicinal agents in commerce globally is unknown, but there are at least 1,000 species alone in China (Duke and Ayensu, 1985; Abas, 2005).

2.2 Cancer

Cancer is the general term for a series of neoplastic diseases that are characterized by changes in a cell leading to abnormal (unordered and uncontrolled) cellular proliferation (Pettit, 1997). The disorder occurs in the normal processes of cell division, which are controlled by the genetic material (DNA) of the cell. Cancers may be caused in one of three ways, namely incorrect diet, genetic predisposition, and via the environment (Reddy et al., 2003).

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10 To date, mortality that results from the common forms of cancer is still unacceptably high. The Second Report of the National Cancer Registry of Malaysia suggested a total of 23,746 cancer cases were diagnosed among Malaysians in the year 2003, comprising 10,473 males and 13,273 females. The crude rate for males was 97.4 per 100,000 population and 127.6 per 100,000 populations for females. The age standardized incidence rate (ASR) for all cancers was 134.3 per 100,000 males and 154.2 per 100,000 females (The Second Report of National Cancer Registry, 2003).

2.2.1 Carcinogens

The majority of human cancers result from exposure to environmental carcinogens; these include both natural and manmade chemicals, radiation and viruses.

Carcinogens may be divided into several classes, such as (i) genotoxic carcinogens, if they react with nucleic acids. These can be directly acting or primary carcinogens, if they are of such reactivity so as to directly affect cellular constituents; (ii) alternatively, they may be procarcinogens that require metabolic activation to induce carcinogenesis;

(iii) epigenetic carcinogens are those that are not genotoxic (Reddy et al., 2003;

Timbrell, 2000). It is also clear that genetic predisposition is one of the factors of human cancers apart from exposure to carcinogens. Thus, patients with the genetic xeroderma pigmentosum are more susceptible to skin cancer (Reddy et al., 2003).

Carcinogens in the diet that trigger the initial stage include moulds and aflatoxins (for example, in peanuts and maize), nitrosamines (in smoked meats and other cured products), rancid fats and cooking oils, alcohol, and additives and preservatives. A combination of foods may have a cumulative effect, and when incorrect diet is added to a polluted environment, smoking, UV radiation, free radicals,

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11 lack of exercise, and stress, the stage is set for DNA damage and cancer progression (Reddy et al., 2003).

2.2.2 Cell cycle

In cells that are dividing, the nuclear DNA molecules must be duplicated and then distributed in a way that ensures the two new cells receive a complete set of genetic instructions. The cells pass through a series of discrete stages called G1 phase, S phase, G2 phase and M phase in order to accomplish these tasks (Kleinsmith, 2006).

These four phases are collectively referred to as the cell cycle.

The cell cycle is commonly represented by a circular diagram (Figure 2.1). The G1, S and G2 phases are collectively referred as interphase. Besides providing the time needed for a cell to make copies of its DNA molecules, interphase is also a period of cell growth. Interphase occupies about 95 % of a typical cell cycle; whereas the actual process of cell division (M phase) only takes about 5 % (Kleinsmith, 2006). G1 is defined as the interval between M phase and S phase, and G2 is defined as the interval between S phase and M phase. S phase is defined as the time during the cell cycle when DNA synthesis is taking place, leading to a doubling of the amount of DNA per cell. M phase is the time when the amount of DNA per cell drops in half as cells divide. The restriction point is a control point near the end of G1 where the cell cycle can be halted until conditions are suitable for progression into S phase. Under normal conditions, the ability to pass through the restriction point is governed mainly by the presence of growth factors.

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12 Figure 2.1: The cell cycle

(Adapted from http://herb4cancer.files.wordpress.com/2007/11/cell-cycle2.jpg, 19 August 2009)

2.2.3 Carcinogenesis

The transformation of a normal cell into a cancerous cell is believed to proceed through many stages over a number of years or even decades. Carcinogenesis is a multistep process that involves initiation, promotion and tumour progression. Initiation involves a reaction between the cancer-producing substance (carcinogen) and the DNA of tissue cells. There may be a genetic susceptibility. This stage may remain dormant, and the subject may only be at risk for developing cancer at a later stage. Promotion involves a prolonged period of proliferation of the initiated cells, occurs very slowly over a period ranging from several months to years. During this stage, a change in diet and lifestyle can have a beneficial effect so that the person may not develop cancer during his or her lifetime. The third and final stage involves progression and spread of the cancer, at which point diet may have less of an impact. Preventing initiation is an important anticancer strategy, as are the opportunities to inhibit cancer throughout the latter stages of malignancy (Reddy et al., 2003; Kleinsmith, 2006).

Restriction point

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13 2.3 Apoptosis in cancer

Most of the cells from higher eukaryotes have the ability to self-destruct by activation of an intrinsic cellular suicide program referred to as programmed cell death or apoptosis (Ellis et al., 1991; Steller, 1995). Apoptosis is an intrinsic biological event that plays an essential role in development, homeostasis and in many disease process.

Culling extra cells in a precise and systematic way is an important aspect of normal development. Hence, the other term for this process is programmed cell death.

Occasionally, cell death program goes awry in several diseases. Degenerative diseases may result in (or be the result of) excessive apoptosis and some cancers appear to inhibit cell death cascades resulting in excessive and uncontrolled proliferation (O’Brien et al., 1998).

Apoptosis is a unique type of cell death, different from what happens when cells are destroyed by physical injury or exposure to certain poisons. In response to such non-specific damage, cells undergo necrosis, a slow type of death in which cells swell and eventually burst, spewing their contents into surrounding tissues. Necrosis often results in a local inflammatory reaction that can cause further cell destruction, which makes it potentially dangerous (Kleinsmith, 2006; O’Brien et al., 1998).

In contrast, apoptosis kills cells quickly and neatly, without causing damage to surrounding tissue. Apoptosis is characterized by certain morphological features, including reduction in cell volume, cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation (Kerr et al., 1972, 1994; Wyllie et al., 1980;

O’Brien et al., 1998). The process involves a carefully orchestrated sequence of intracellular events that systematically dismantle the cell (Figure 2.2).

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14 Figure 2.2: Main steps in apoptosis (Kleinsmith, 2006)

As a cell begins to undergo apoptosis, the first observable change is cell shrinkage. Next, small bubble-like protrusions of cytoplasm form at the cell membrane as the nucleus and other cellular structures begin to disintegrate. The chromosomal DNA is then degraded into small pieces and the entire cell breaks apart, forming small fragments known as membrane-bound apoptotic bodies that are engulfed by macrophages or neighbouring phagocytic cells without generating an inflammatory response. Phagocytic cells are specialized for ingesting foreign matter and breaking it down into molecules that can be recycled for other purposes (Kleinsmith, 2006).

The genetic basis for apoptosis implies that cell death, like any other metabolic or development program, can be disrupted by mutation. Elucidation of the core machinery of apoptosis has provided new insights into cancer biology, revealing novel strategies for cancer therapy (Reed, 2002). In the last decade, basic cancer research has produced remarkable advances in the understanding of cancer biology and cancer genetics. Among the most important of these advances is the realization that apoptosis and the genes that control it have a profound effect on the malignant phenotype. For example, it is now clear that some oncogenic mutations disrupt apoptosis, leading to tumour initiation, progression or metastasis. Conversely, compelling evidence indicates

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